KINETICS AND MECHANISM OF MEMBRANE INTERACTIONS WITH

ANTIMICROBIAL PEPTIDE ANALOGS OF CECROPIN A AND MAGAININ 2

Kim S. Clark

A Thesis Submitted to the

University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree of

Master of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2010

Approved by

Advisory Committee

Richard M. Dillaman S. Bart Jones

Paulo F. Almeida

Chair

Accepted by

Dean, Graduate School

ii

This thesis has been prepared in the style and format

consistent with the journal

Biochemistry

iii

TABLE OF CONTENTS

ABSTRACT .....................................................................................................................................v

ACKNOWLEDGEMENTS ........................................................................................................... vi

DEDICATION .............................................................................................................................. vii

LIST OF TABLES ....................................................................................................................... viii

LIST OF FIGURES ....................................................................................................................... ix

INTRODUCTION ...........................................................................................................................1

MATERIALS AND METHODS ...................................................................................................13

Chemicals ...........................................................................................................................13

Peptides ..............................................................................................................................15

Buffer Preparation ..............................................................................................................15

Large Unilamellar Vesicle Preparation ..............................................................................16

Lipid Concentration Determination ...................................................................................16

7-Methoxycoumarin Lipid Probe Synthesis ......................................................................17

Fluorescence Experiments .................................................................................................18

Circular Dichroism Experiments .......................................................................................23

RESULTS ......................................................................................................................................24

Kinetic Binding Experiments .............................................................................................24

Dye Efflux Experiments ....................................................................................................36

ANTS/DPX Assay .............................................................................................................41

Circular Dichroism Experiments .......................................................................................45

Thermodynamics of Peptide-Lipid Interactions ................................................................48

iv

DISCUSSION ................................................................................................................................51

FUTURE WORK ...........................................................................................................................55

ABBREVIATIONS .......................................................................................................................56

REFERENCES ..............................................................................................................................57

v

ABSTRACT

There are many factors which control the interactions of a peptide and membrane. The

kinetics and mechanism of these interactions were investigated for variants of cecropin A and

magainin 2. In these variants, amino acid residues were mutated in an attempt to conserve the

properties of the parent peptide while minimizing variety in the variant peptide’s sequence. Cell

membranes were modeled with large unilamellar vesicles composed of various neutral and

negatively charged diacyl phospholipids. Fluorescence experiments were performed to measure

binding kinetics and dye efflux, and the ANTS/DPX assay was used to determine the membrane

perturbation mechanism. Peptide helicity was analyzed using circular dichroism. The binding

kinetics were in line with our estimates, and the efflux levels of the parent peptide and the variant

peptides were the same. However, the mechanism of membrane perturbation changed in both

cases, from all-or-none in the parent peptides, to graded in the variants.

vi

ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude to Dr. Paulo Almeida, whose support, advice, and

guidance made this work possible. Muito obrigado.

I would like to thank Dr. Antje Almeida, whose insight and perspective helped shape mine and

open my eyes to the different view of stuff. Vielen Dank.

I would like to thank Dr. S. Bart Jones and Dr. Richard Dillaman for their patience and feedback

on my advisory committee.

I would like to thank Laura Huskins, Sterling Wheaten, Erin Kilelee, Sarah Higgins, Jeff Naro,

Alicia McKeown, Alex Kreutzberger, Sarah Pagentine, and Julia Nepper for their assistance in

techniques, letting me bounce ideas off of them, washing dishes, for being such great labmates,

and making my time in the Almeida lab thoroughly enjoyable.

The Department of Chemistry, the United States Army Reserve and the National Institutes of

Health provided financial support for my research and studies.

vii

DEDICATION

I would like to dedicate this thesis to my wife: Sherri, you are the love of my life and the reason

I live.

viii

LIST OF TABLES

Table Page

1. One-Letter Amino Acid Sequences of the Native and Modified Peptides ..........................5

2. Cecropin A and CE2 On- and Off-Rate Constants and

Equilibrium Dissociation Constants ..................................................................................30

3. Magainin 2 and MG2 On- and Off-Rate Constants and

Equilibrium Dissociation Constants ..................................................................................35

4. Thermodynamic Parameters for Peptide Binding and Insertion into POPC

Bilayers at Room Temperature ..........................................................................................50

ix

LIST OF FIGURES

Figure Page

1. Major Structural Classes of Antimicrobial Peptides ............................................................4

2. The All-or-None and Graded Release Mechanisms ............................................................7

3. Helical Wheel Projections of Cecropin A and CE2 Peptides at Neutral pH .......................9

4. Helical Wheel Projections of Magainin 2 and MG2 Peptides at Neutral pH ....................11

5. Structure of POPC, POPG, and POPE ...............................................................................14

6. Absorption and Emission Spectra of Tryptophan and 7-Methoxycoumarin .....................19

7. Schematic of Fluorescence Resonance Energy Transfer from Tryptophan to

7-Methoxycoumarin ...........................................................................................................20

8. Lipid Concentration Effects on Peptide Binding ...............................................................25

9. Kinetics of CE2 Binding to Various Compositions of POPC/POPG Vesicles ..................27

10. CE2 On- and Off-Rates as a Function of POPC Content in Mixed

POPC/POPG Vesicles ........................................................................................................28

11. CE2 Equilibrium Dissociation Constants as a Function of POPC

Content in POPC/POPG Mixed Vesicles ..........................................................................29

12. Kinetics of MG2 Binding to Various Compositions of POPC/POPG Vesicles ................32

13. MG2 On- and Off-Rates as a Function of POPC Content in Mixed

POPC/POPG Vesicles ........................................................................................................33

14. MG2 Equilibrium Dissociation Constant as a Function of POPC

Content in POPC/POPG Mixed Vesicles ..........................................................................34

15. CE2 Dye Efflux Experiments at Various Concentrations of 50:50

POPC/POPG Vesicles ........................................................................................................37

16. MG2 Dye Efflux Experiments at Various Concentrations of 50:50

and 70:30 POPC/POPG Vesicles .......................................................................................39

17. MG2 Dye Efflux Experiment with 50 µM 100% POPC Vesicles .....................................40

18. ANTS/DPX Assay for CE2 ...............................................................................................43

x

19. ANTS/DPX Assay for MG2 ..............................................................................................44

20. CD Spectra for CE2 with 50:50 POPC/POPG and 100% POPC.......................................46

21. CD Spectra for MG2 with 50:50 POPC/POPG and 100% POPC .....................................47

INTRODUCTION

There has been a vast amount of research on antimicrobial peptides since their discovery

three decades ago (1). These are small, endogenous peptides characterized by their ability to

bind and neutralize various infectious microorganisms (2). Antimicrobial peptides have an

uncanny ability to efficiently discriminate between self and non-self which reasonably justifies

their presence in all kingdoms of life (3). The means by which they defeat foreign cellular

bodies arises from the nature of the peptides themselves. These peptides in general contain

between 10 and 50 amino acids, are basic and positively charged at biological pH, and are

amphipathic. These peptides vary in several properties which contribute to their unique

interactions with cell membranes, and hence their activity: sequence, size, structure, charge,

hydrophobicity, and amphipathicity (4). Variability in these characteristics generates several

results: it allows an organism to produce peptides which can respond to an assortment of

different invading microbes based on their cell type and allows several peptides to work

synergistically to overcome infection by departmentalizing their functions. The latter may take

place because some peptides function by direct interaction with and disruption of the membrane.

Others permeate the membrane of a foreign microorganism and interact with internal

components of that cellular system (5).

The sequence of the peptides determines the interactions that it will have with potential

targets as well as what secondary conformation the peptide can adopt. In turn, the secondary

structure determines how the amino acid residues arrange themselves spatially, contributing to

hydrophobicity and amphipathicity. The hydrophobic character determines how easily the

peptide can partition from the biological matrix to the membrane. The amphipathicity is the

arrangement of the nonpolar hydrophobic residues opposite from positively charged basic

2

residues. Many peptides adopt this arrangement upon interacting with a target membrane (6).

The size of the peptides determines their area and thus defines the potential facial interaction, as

well as other physical properties of the peptide such as overall charge and charge distribution.

As with all biological systems, there is a dynamic relationship between a peptide, its

biological transport system (solvent), and its specified target (membrane surface). Most

eukaryotic organisms’ primary defense against bacteria, fungi, and viruses is attributed to

cationic antimicrobial peptides (5,7). Current work also probes the feasibility of using certain

peptides as anticancer treatments (8).

As the first tier defense, these peptides must be nonspecific for invading bodies and either

eliminate the infection or provide sufficient time for the host adaptive immune system to be

mobilized. With these requirements, these peptides are often found in the epithelial layers and

within phagocytic cells in larger organisms (9). The short sequences of amino acids facilitate

simple and rapid synthesis, whether the assembly machinery is biological, in vivo, or synthetic,

in vitro (4). The evolutionary success of antimicrobial peptides is demonstrated in a cell’s ability

to respond quickly to infectious agents and evidenced by their occurrence in a variety of species.

With the increase in resistance due to widespread misuse and overuse of current classical

antibiotic treatments, alternate means of treating infections must be explored.

Though antimicrobial peptides are sequentially, synthetically, and structurally simpler

than proteins, the exact correlation between structure, function, and activity is yet elusive; there

probably is no direct correlation between them (10). It is our hope that this research will shed

some light in this area. This study attempts to reinforce established relationships between these

properties to better understand how various antimicrobial peptides affect permeabilization and

3

liquidity of cells. More precisely, we hope to show that the specific amino acid sequence is not

important by itself, but that the properties of the amino acids at specific locations in the primary

sequence affects overall peptide action. The kinetics and mode of action of peptide-lipid

interactions were compared to the native peptide by modifying the primary sequence and

replacing several amino acid residues with a ―minimalist‖ analog. It was our intent to preserve

the secondary structure of the antimicrobial peptides, and gauge the effects caused by

conservatively modifying targeted individual residues.

There are several structural classes of antimicrobial peptides. The major structure types

include α-helical, β-stranded, extended coil, and loops (11). Examples of these can be seen in

Figure 1. The peptides in this study are solely the α-helical type: cecropin A, and magainin 2.

We wanted to test the hypothesis that mutations of most residues in a peptide are neutral,

attributing minimal change to the overall peptide properties. In our studies, these two peptides

were modified into WAL analogs, composed of a tryptophan, and many alanine and leucine

residues. These are ―minimalist‖ versions of the peptides where Leu (L) replaced all

hydrophobic residues and Ala (A) replaced all others. The intrinsic fluorophore Trp (W) was

retained when included in the original sequence, or was added. The residues Gly, Pro, Asp, Glu,

and Lys were maintained to conserve charge, except that Arg was replaced by Lys. Some

additional residues were modified to either retain conformational requirements of the peptide

sequence, or to keep thermodynamic values close to the values of the native peptides. The major

differences between the peptides arise in the kinetics of peptide-membrane interaction, the

energy required for insertion into the lipid bilayer, and the efflux kinetics of lipid contents. We

expected that the neutrality of the mutations would produce results that are indistinguishable

between the native and WAL mutant of the peptides, the detailed sequences of which are shown

in Table 1. The cecropin A mutant is called CE2 and the magainin 2 mutant is MG2.

4

Figure 1: Major structural classes of antimicrobial peptides. The yellow colored ribbons represent β-sheets, and the

magenta colored portions in (C) represents an α-helix and in (D) represents the loop portion. (A) Extended

indolicidin (PDB ID 1G89); (B) β-stranded hepcidin (PDB ID 1M4E); (C) α-helical magainin 2 (PDB ID MAG2);

(D) looped thanatin (PDB ID 8TFV). These were modeled using the RasMol molecular graphics program, v.2.7.5

(12).

C

A B

D

5

Table 1: One-Letter Amino Acid Sequences of the Native and Modified Peptides. The gray boxes highlight

mutations from the original sequence to the variant.

Peptide Sequence

Cecropin A KWKLF KKIEK VGQNI RDGII KAGPA VAVVG QATQI AK-amide

CE2 KWKLL KKLEK AGAAL KEGLL KAGPA LALLG AAAAL AK-amide

Magainin 2 GIGKF LHSAK KFGKA FVGEI MNS

MG2 GLGKL LHAAK KLGKA WLGEL LAA

6

The means by which the antimicrobial peptides perturb a membrane can vary, but can be

classified as either all-or-none or graded. More popular mechanisms include the barrel-stave

model, the toroidal pore model, the carpet model, the sinking raft model, and other models

describing less structured pores (13). In the all-or-none model, individual vesicles either

completely release their contents or they release nothing at all. In the graded model, all vesicles

release the same portion of their contents. A simplified diagram representing the two extremes

of the all-or-none and graded mechanisms of release is shown in Figure 2.

The all-or-none release mechanism is normally attributed to the barrel-stave model, the

carpet model, and in some instances, the toroidal pore. In this mechanism, the Gibbs free energy

of insertion of the peptide from the membrane interface to the hydrophobic core of the bilayer is

hypothesized to be larger than 20 kcal/mol (10). This mechanism consists of four different

states: 1) unstructured, unbound peptide free in solutions; 2) peptide bound as an α-helix to full

vesicles; 3) peptide inserted into the lipid bilayer in a pore-state; and 4) peptide bound to empty

vesicles (14).

The following are several models which display the necessary criteria for the all-or-none

mechanism. In the barrel-stave model, several peptides insert into the membrane perpendicular

to the bilayer forming a pore—the peptides, in contact with each other, form the ―staves‖ and the

overall structure provides the ―barrel‖ shape (15). The hydrophobic portions of the peptides

point toward the acyl chains of the lipids and the hydrophilic regions line the solution side of the

pore, allowing cytoplasmic contents to easily cross to the extracellular region and also causing

potential loss of electrochemical gradients. The carpet model involves peptides orienting

themselves parallel to the lipid bilayer, and upon reaching a critical threshold, they cause

permeabilization (6,9). They do this by coating the membrane like a carpet, which requires

7

Figure 2: The all-or-none and graded release mechanisms. All-or-none release of 50% of the vesicles causes half of

the vesicles to release all of their contents and the other half to remain fully intact. Graded release of 50% of the

vesicles contents causes all vesicles to lose half of their contents. The details of the different mechanisms were

omitted from this schematic drawing, but are discussed in detail by Yandek et al. (14).

8

relatively high concentrations of peptides compared to other models. Since the concentration of

peptides in this model is high, the action of the peptides is detergent-like and can cause

micellization. The toroidal pore model (16,17) involves several peptides inserting

perpendicularly into the membrane, however, unlike the barrel-stave model, they do not

necessarily have to be in contact with each other. Portions of the membrane can fold in and fill

the space between the peptides, creating peptide and lipid lined pores.

Graded release is attributed to the toroidal pore and sinking raft mechanisms of

antibacterial peptide action. However, for toroidal pores to invoke graded release, the pore state

must be short-lived. In the sinking raft model, a stochastic structure of peptides complex and

form the pore (18-20). The structure formed in this model can have the peptides insert into the

membrane either perpendicular, parallel, or both. The mechanism of release is determined by the

type of peptide as well as the free energy of insertion. It is our working hypothesis that if the

free energy of insertion is greater than 20 kcal/mol, then the release will be all-or-none, and if it

is below this threshold, it will be graded.

Cecropin A is a 37 amino acid peptide derived from the giant silk moth, Hyalophora

cecropia (21,22). Upon binding, cecropin A adopts a secondary structure with two alpha-helical

regions, one from Phe5 to Lys21 and the other from Pro24 to Gln37, linked by a Gly-Pro break

(22,23). These two regions can be seen in a helical wheel projection in Figure 3. In this

projection it is easy to see the amphipathicity that the peptide can adopt as a helix. Also visible

in comparing the native and minimalist WAL versions of the peptide is the conservation of the

residue properties at each location. The only noted change in individual amino acid properties is

seen at position 11: the mutation Ala11 Val11, going from an uncharged polar residue to a

nonpolar residue.

9

Figure 3: Helical wheel projections of cecropin A and CE2 peptides at neutral pH. A and B are projections of

cecropin A. C and D are projections of CE2. A and C represent the first helical segments of the peptide, composed

of residues 5 to 21. B and D represent the second helical segments, residues 24 to 37. Blue symbols are basic,

positively charged residues. Red symbols are acidic, negatively charged residues. White symbols are polar but

uncharged residues. Gray symbols are nonpolar residues.

C A

D B

10

Magainin 2 is a 23 residue peptide originally extracted from the skin of the African

clawed tree frog, Xenopus laevis. When discovered, it was aptly named from the Hebrew word,

―magain‖, meaning ―shield‖ (24). Magainin 2 adopts a single helix conformation upon binding.

The helical wheel projections of Magainin 2 and MG2 can be seen in Figure 4. Experiments

show that overall, magainin 2 invokes all-or-none release with data that suggests the mechanism

is either toroidal pore formation or the sinking raft model (25).

Large unilamellar vesicles (LUVs) were used to model bacterial cell membranes in these

experiments. For some experiments, incorporation of a fluorophore was necessary. The

concentrations of diacylphosphatidylglycerol (POPG) and diacylphosphatidylcholine (POPC)

lipids were varied based on previous work with the respective peptides to test the similarity of

effectiveness of the WAL mutants and the native peptides. These model the polar head groups

of lipids found in microorganisms – phosphatidylglycerol being anionic and phosphatidylcholine

being neutral. Varying the compositions of the lipid vesicles allows production of LUVs which

have cell membrane characteristics, simulating native peptide binding conditions.

The kinetics of peptide binding will be calculated for the WAL mutants and compared to

the native peptides using fluorescence energy transfer experiments. Each peptide being studied

has a Trp residue which is used to transfer energy upon excitation to a fluorophore, 7-

methoxycoumarin (7MC), which is incorporated into the lipid vesicles by attachment to the

phosphate headgroup of the lipid diacylphosphatidylethanolamine (POPE). The 7MC is a probe

that indicates the proximity of the peptide to the vesicle. The neutral residue mutations that we

are investigating should impart little or no change in the binding constants previously determined

for the native peptides.

11

Figure 4: Helical wheel projections of magainin 2 (A) and MG2 (B) at neutral pH. The helical portion of these

peptides includes the entire sequence. Blue symbols are basic, positively charged residues. Red symbols are acidic,

negatively charged residues. White symbols are polar but uncharged residues. Gray symbols are nonpolar residues.

B

A

12

MG2 has a calculated Gibbs free energy of insertion of 25 kcal/mol, based on an

estimated helicity of 70%. We expected that this peptide should release vesicle contents by an

all-or-none mechanism. Since CE2 has calculated insertion energy of 36 kcal/mol with an

assumed helicity of 70%, all-or-none release was also expected with this peptide. One caveat to

consider is the helicity of the peptide in free solution and the helicity of the peptide in the bound

state. In the original hypothesis proposal, calculations were performed using an assumed helicity

of 70% in the bound state and minimal helicity free in solution. However, the experimentally

determined helicities are different than this and affect the values obtained for the free energy of

insertion and the thermodynamic values calculated previously.

Incorporation of a fluorescent dye within the LUVs facilitates studies of the efflux

kinetics of the vesicle in the presence of peptide. Initially at self-quenching concentrations

(50mM), carboxyfluoroscein dye cannot fluoresce while still encapsulated in the LUV. Once the

membrane is perturbed, dye released into external buffer is diluted and can fluoresce. This

allows kinetics and total release levels to be calculated.

13

MATERIALS AND METHODS

Chemicals

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-

glycero-3-phospho-(1’-rac-glycerol)(sodium salt) (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-

3-phosphoethanolamine (POPE) were all purchased in chloroform solution from Avanti Polar

Lipids, Inc. (Alabaster, AL) (see Figure 5). 7-methoxycoumarin-3-carboxylic acid, succinimidyl

ester (7MC), 5-(and -6)-carboxyfluorescein (CF), 8-aminonaphthalene-1,3,6-trisulfonic acid,

(ANTS), and p-xylene-bis-pyridinium bromide (DPX) were purchased from Molecular

Probes/invitrogen™ (Eugene, OR). 3-morpholinopropane-1-sulfonic acid (MOPS), ULTROL®

grade, was purchased from EMD Chemicals (Gibbstown, NJ). Potassium chloride (KCl),

potassium hydroxide (KOH), ethylenediaminetetraacetic acid (EDTA), and sodium azide (NaN3)

were all purchased from BDH (West Chester, PA). Ethanol (EtOH), 200 proof, was purchased

from AAPER Alcohol and Chemical (Shelbyville, KY). Dichloromethane (DCM), methanol

(MeOH), and all other organic solvents were HPLC or ACS grade and purchased from Burdick

& Jackson (Muskegon, MI). Ammonium molybdate, ACS grade, was purchased from Thermo

Fischer Scientific (Fairlawn, NJ). Dimethylformamide (DMF), ascorbic acid (USP grade) and

perchloric acid (70%, ACS grade), were purchased from Mallinckrodt Chemicals (Phillipsburg,

NJ). Water, filtered to 18.2 MΩ∙cm purity using a Milli-Q Direct Water Purification System by

Millipore (Billerica, MA), was obtained on site and stored in a 20 L Nalgene® carboy.

14

Figure 5: Structure of POPC, POPG, and POPE. The only difference between these phospholipids appears in the

headgroup. Neutral, zwitterionic POPC is shown (top). Negatively charged POPG is shown as a sodium salt

(middle). Neutral POPE, a reactant in the 7MC-POPE synthesis is also shown (bottom).

15

Peptides

CE2 (KWKLLKKLEKAGAALKEGLLKAGPALALLGAAAALAK-amide) lot:

B05973, >82% purity, was purchased from Bachem, Inc. (Torrance, CA). CE2, lot: pr1770, 98%

purity, and MG2 (GLGKLLHAAKKLGKAWLGELLAA), lot: pr1202, 94% purity, were

purchased from New England Peptide, LLC (Gardner, MA). Lyophilized peptide was stored at

-20°C. Stock peptide solutions were prepared by mixing lyophilized peptide in 1:1 (v/v)

water/ethanol. Peptide concentration was determined using a Cary 1E UV-Vis

spectrophotometer, in matched 1.000 cm 6Q quartz cells, scanning from 500 to 250 nm using the

absorbance maximum of tryptophan at 280 nm with an extinction coefficient of 5,579 M-1

cm-1

.

Solutions were then aliquoted into small Eppendorf™ tubes and flash frozen using either liquid

N2 or an acetone/dry ice bath. Peptide solutions were stored at -80°C and kept on ice during

experiments.

Buffer Preparation

MOPS buffer was prepared in water with 20mM MOPS, 100 mM KCl, 0.1 mM EDTA

and 0.02% NaN3, then brought to pH 7.50 using 1 M KOH and brought to final volume. CF

buffer was prepared by grinding CF powder with mortar and pestle, then mixing 50 mM CF, 20

mM MOPS, 0.1 mM EDTA, and 0.02% NaN3, in water and brought to pH 7.50 with 1 M KOH,

then brought to final volume. ANTS/DPX hydration buffer was prepared in water with 5mM

ANTS, 10mM DPX, 20mM MOPS, 80mM KCL, 1.0 mM EDTA, and 0.02% NaN3.

ANTS/DPX titration buffer was prepared in water with 45 mM DPX, 20 mM MOPS, 30 mM

KCl, 1.0 mM EDTA, and 0.02% NaN3.

16

Large Unilamellar Vesicle Preparation

All glassware and syringes were cleaned by rinsing or vortexing with 1:1 DCM/MeOH.

Large unilamellar vesicles (LUVs) were prepared by combining appropriate volumes of stock

lipid solutions in a round bottom flask. The solvent was rapidly removed using a rotary

evaporator at 65°C (Buchi R-3000, Flawil, Switzerland). The lipid film was then dried for at

least 4 hours in a dessicator under vacuum with ~100g Drierite™ in the base. The lipid film was

then hydrated with the appropriate buffer and vortexed 1 minute to remove all lipids from the

glass walls and suspend them. The lipid solution was then transferred to a test tube and

subjected to five freeze-thaw cycles using liquid N2 (or a dry ice/acetone bath), then room

temperature water, then ~40°C water. This was done to promote vesicle fusion, creating large

multilamellar vesicles, and assisting dye encapsulation (for CF and ANTS/DPX vesicles). A

high-pressure extruder (10 mL water-jacketed Lipex Extruder, Lipex Biomembranes, Vancouver,

CAN) was assembled with two stacked Nucleopore 0.1 µm polycarbonate filters (Whatman,

Florham, NJ), and rinsed once with 10 mL MOPS buffer, then ten times with 1.5 mL MOPS

buffer each. The lipid solution was then extruded ten times at 450 psi, to create homogeneous,

100 nm unilamellar vesicles. CF and ANTS/DPX vesicles were subsequently passed through a

Sephadex® G-25 column to remove unencapsulated dye.

Lipid Concentration Determination

A modified Bartlett assay was used to determine the phosphate concentration of the lipid

solutions and hence, the lipid concentration (28). Briefly, 1.053 mM phosphate standard was

pipetted in 60 µL increments from 0 to 300 µL along with vesicle solution samples in glass test

tubes. All were pipetted in triplicate with the balance as water to keep volumes equal. 700 µL of

17

70% perchloric acid was added and the solutions were heated under reflux at 210°C in an

aluminum heating block for 1 h, to cleave phosphate headgroups from the phospholipids. This

was allowed to cool, then 2.00 mL of 1.0% (w/v) ammonium molybdate/water solution was

added to each tube and vortexed for 1 s. Next, 2.00 mL of 4.0% (w/v) ascorbic acid solution was

added to each tube and vortexed again for 1 s. The tubes were then placed in a 37°C water bath

for 45 minutes, allowing formation of the phosphomolybdate complex. The absorbance was then

read at 580 nm using a spectrophotometer (Spectronic 20D, Thermo Scientific, Waltham, MA).

The unknown lipid concentration was determined using the linear regression of the phosphate

standards.

7-Methoxycoumarin Lipid Probe Synthesis

7MC-POPE (7MC amide linked to the POPE headgroup) was synthesized using the Vaz

and Hallman method (29). Briefly, all organic solvents were dried for 24 h using molecular

sieves (Sigma-Aldrich, St. Louis, MO). Approximately 700 µL stock POPE stored in CHCl3

was dried with rotary evaporation then redissolved in 700 µL dry CHCl3. Next, 10 mg 7MC was

dissolved in dry DMF and added to an aluminum foil-wrapped test tube, followed by 2.3mg of

ground, dry K2CO3. The 700 µL POPE (of ~[60 µM]) solution was then added to the mixture

which was stirred in the dark until the reaction was complete (usually less than 2 h). The

reaction was monitored by SiO2 TLC using 65:25:4 (v/v/v) CH2Cl2/MeOH/water. The 7MC was

visualized by UV lamp, and the lipid by Zinzade’s reagent followed by ashing (30). The product

was purified by either preparatory SiO2 TLC or SiO2 column using 2:1 (v/v) CH2Cl2/MeOH.

7MC-POPE was then dried using rotary evaporation then redissolved in a minimal volume of dry

CHCl3. Concentration of the lipid probe was determined using a spectrophotometer (Cary 1E

18

UV-Vis, Varian, St. Helens, Australia), using the absorption maximum at 348 nm and ε =

2.9×104 M

-1 cm

-1.

Fluorescence Experiments

Kinetic Binding Experiments. Binding kinetics for CE2, MG2, and TP2 were determined

using an SX.18MV-R stopped-flow fluorometer (Applied Photophysics, Leatherhead, United

Kingdom). Binding vesicles were created using various compositions of POPC and POPG, also

including 2 mol% 7MC-POPE. Excitation was set at 280 nm and emission was monitored

through a 385 nm long-pass filter by Edmund Industrial Optics (Barrington, NJ). This emission

wavelength was chosen because the emission maximum of 7MC-POPE at 396 nm, is the same as

7MC. The overlap of the tryptophan emission and 7MC absorption is shown in Figure 6, and a

schematic of the energy transfer is shown in Figure 7. Experiment concentrations were always 1

µM peptide and lipid concentration normally varied between 25 and 200 µM.

19

Figure 6: Absorption and emission spectra of tryptophan and 7MC. Overlap of the emission of tryptophan and the

absorption of 7MC is the necessary condition which allows FRET to occur.

20

Figure 7: Schematic of fluorescence resonance energy transfer from tryptophan to 7-methoxycoumarin. When the

intrinsic tryptophan is greater than the Förster distance from 7MC, no energy transfer can occur (left). As the

peptide binds and interacts with the membrane, energy transfer to the 7MC occurs and can be monitored (right).

21

Analysis of Binding. Binding analysis was performed based on the work of Gregory et al.

(13,25). Briefly, the reversible binding of a peptide-lipid complex can be described by the

equilibrium expression, Eq. 1.

(1)

Here P is peptide, L is lipid, PL is the peptide-lipid complex, kon is the on-rate constant and koff

is the off-rate constant. The peptide-lipid complex formation can be described as a differential

function of time as seen in Eq. 2:

(2)

Here, the bracketed terms represent concentrations of the lipid, peptide and peptide-lipid

complex. Assuming [P] << [L], and that the lipid concentration is constant (lipids are not

consumed in these analyses), the rate law described is pseudo 1st order. The data were fit with

the time-dependent solution to Eq. 2, a one-exponential curve, described as Eq. 3:

(3)

These experiments are conducted in non-equilibrium conditions and hence the apparent rate

constants, kapp, were calculated using Eq. 3. The constants C1 and C2 correspond to the intensity

(amplitude) and fluorescence signal value at time = 0 (y-intercept), respectively. The apparent

rate constant can be expressed as the linear relationship in Eq. 4:

(4)

and the equilibrium dissociation constant, KD, written as:

22

. (5)

Carboxyfluoroscein Efflux Experiments. LUVs were prepared by hydrating mixed

compositions of POPC/POPG lipid films using CF buffer. Fluorescence was monitored over

time using an SX.18MV-R stopped-flow fluorometer (Applied Photophysics, Leatherhead,

United Kingdom). As peptides perturb the vesicle, the self-quenching encapsulated CF is

released into the surrounding buffer, and is diluted, leading to an increase in fluorescence signal.

Excitation was set at 470 nm and emission measured through a 530 nm long-pass filter (Edmund

Industrial Optics, Barrington, NJ). Peptide concentration was always 1 µM and lipid

concentration was varied from 25 to 200 µM. The fractional release was normalized using the

maximum fluorescence levels determined by dissolving the vesicles using 1% Triton X-100,

comparing fluorescence of the solution before and after complete vesicle disruption. Due to

error and variances in technique and instrumentation, ―full release‖ is loosely defined as when a

peptide induces fractional CF release between ~80 and 100%.

ANTS/DPX Assay. The ANTS/DPX assay was performed to discover the mechanism of

peptide action, as described in detail by Ladohkin et al. (26,27). LUVs were prepared by

hydrating mixed compositions of POPC/POPG lipid films using ANTS/DPX hydration buffer.

Fluorescence measurements were recorded in an SLM-Aminco 8100 spectrofluorimeter

upgraded with monochromator stepper and photon counter modules by ISIS. Excitation was set

at 365 nm, with 1 nm slit width, and emission monitored at 515 nm, using an 8 nm slit width.

Vesicle concentration was constant in the experiments, and estimated at 600 µM. Varying

volumes of peptide solution were added depending on the concentration of the solution, but

usually in the range of 1 to 30 µL.

23

Circular Dichroism Experiments

Circular dichroism experiments were performed using a Chirascan (Applied

Photophysics, Leatherhead, United Kingdom). The secondary structure of peptides was

calculated both free in solution and bound to various composition vesicles. Experiments were

performed in a 1 mm quartz cell at 25°C, using 1 nm steps from 190 to 260 nm and corrected for

background signal from buffer and vesicles. The estimated peptide helicities were determined

using the spectral minimum at 222 nm (31):

(6)

where [Θ222] is the molar ellipticity at 222 nm, expressed as deg∙cm2∙dmol

-1 (32), and [Θcoil] and

[Θhelix] are given by Eqs. 7 and 8 (33):

(7)

(8)

The parameter n is the number of amino acids in the peptide sequence and T represents

temperature in °C.

24

RESULTS

Kinetic Binding Experiments

Binding of CE2 and MG2 was measured by fluorescence resonance energy transfer

(FRET) between the tryptophan incorporated in all of these peptide sequences and 7MC-POPE.

Excitation of tryptophan at 280 nm causes fluorescence at 350 nm. The tryptophan of a peptide

within the Förster distance to a 7MC labeled phospholipid incorporated in the lipid membrane,

can transfer this energy to 7MC, consequently fluorescing at 396 nm (see Figures 6 and 7).

Since this energy transfer is a function of distance between the FRET pair and only occurs when

both are about 28Å apart or less, increase in fluorescence at 396 nm is indicative of the binding

event.

The binding data were fit with Eq. 3, and kapp was plotted as a function of lipid

concentration. Linear regression of these plots gave kon (the slope) and koff (the y-intercept) via

Eq. 4, further allowing calculation of KD. Modification of regression fits was sometimes

necessary to account for deviation in certain constants. If the y-intercept is especially close to

the origin, significant error in the experimental value for koff may exist. If this is the case,

dissociation experiments should be performed to establish an accurate value for the off-rate

constant.

At high peptide to lipid ratios, the kinetics of binding deviate from linearity. The

numerous peptides per vesicle tend to disrupt the vesicle catastrophically, causing micellization

or destroying the integrity of the vesicle and the lipid bilayer. Since peptide concentration is

held constant, this occurs near the lower lipid concentration range in these experiments, around

25 µM and below. At such low lipid concentrations, the peptide:vesicle ratio can approach

5000:1 and the results can be seen in Figure 8.

25

Figure 8: Lipid concentration effects on binding. This plot of kapp as a function of lipid concentration for CE2

shows that as the lipid goes below ~30 µM, the curve deviates from linearity. The interactions between the peptides

and vesicles may lead to micellization or other catastrophic events for the vesicles, affecting the binding constants at

low lipid concentration. The solid line is the linear fit for all points, the dashed line is fit to the region 30 to 200 µM,

and the dotted line is fit from 5 to 30 µM.

26

CE2 Binding

CE2 binding to pure POPC vesicles was marginal, thus binding had to be performed on

various compositions of vesicles in order to determine binding constants for pure POPC. Vesicle

compositions of 50:50:2 up to 90:10:2 POPC/POPG/7MC-POPE were used. A representative

example of the data collected in these experiments is shown in Figure 9. Various graphs of KD,

kon, and koff as a function of vesicle POPC content were used to extrapolate values for pure POPC

vesicles, and are seen in Figures 10 and 11. The binding kinetic constants and dissociation

constants are compared for cecropin A and CE2 in Table 2.

27

Figure 9: Kinetics of CE2 binding to vesicles of POPC/POPG 50:50 (A and B), 70:30 (C and D), 80:20 (E and F),

and 90:10 (G and H). On the left, the curves represent ~20 averaged experimental binding kinetics traces at 25 µM

lipid and 1 µM CE2, and the curve is the single-exponential fit to the data (Eq. 3). To the right of each graph is the

apparent rate constant plotted as a function of lipid concentration, yielding koff from the y-intercept and kon from the

slope (Eq. 4)

28

Figure 10: (A) CE2 on-rates as a function of POPC content in mixed POPC/POPG vesicles. The binding

experiments were performed with 1µM CE2 and various lipid concentrations from 25 to 200 µM. Increasing the

neutral character of vesicles causes marked decrease in the affinity of the peptide for the vesicle. When the

composition reaches a certain point, around 80 to 90%POPC, the on-rate is greatly affected and deviates from the

other data. (B) CE2 off-rates as a function of POPC content in mixed POPC/POPG vesicles. The off-rate at 100%

POPC can be calculated based on a fit of the data through 80% POPC. Above this point, binding is weak, and the

off-rate is artificially deflated, seen at 90% POPC.

29

Figure 11: (A) CE2 equilibrium dissociation constant as a function of POPC content in POPC/POPG mixed

vesicles. The exponential fit is weighted based on the variance of the KD values and shows the extrapolation at

100% POPC. (B) The natural logarithm of the equilibrium dissociation constant as a function of POPC content in

POPC/POPG mixed vesicles. Again, the regression can be used to extrapolate the 100% POPC value for KD. The

deviation of koff at high POPC percentage leads to deflated KD values, seen in the 90% value.

30

Table 2: Cecropin A and CE2 On- and Off-Rate Constants and Equilibrium Dissociation Constants

Vesicle composition Peptide Binding kinetics Dissociation constant*

POPC/POPG kon (M-1

s-1

) koff (s-1

) KD (µM)

50:50 Cecropin A

CE2 (5.2 ± 0.2) 10

5

(6.4 ± 0.1) 105

7 ± 2

15 ± 2

13 ± 4

23 ± 3

70:30 Cecropin A

CE2 (4.3 ± 0.4) 10

5

(2.3 ± 0.2) 105

46 ± 4

86 ± 3

110 ± 14

377 ± 35

80:20 Cecropin A

CE2 (3.9 ± 4.0) 10

5

(1.7 ± 0.7) 105

170 ± 40

100 ± 10

440 ± 460

700 ± 300

90:10

100:0‡

Cecropin A

CE2

Cecropin A

CE2

ND†

(8 ± 2) 104

2 105

9.4 104

ND

81 ± 3

300

370

—

1100 ± 280

1000

5000

*KD is calculated solely from binding kinetics and expressed as a function of lipid concentration, not vesicle

concentration. †These values were not determinable due to weak signal. ‡Values for 100% POPC vesicles were

extrapolated from the best-fit curves for cecropin A and CE2.

31

MG2 Binding

MG2 binding to pure POPC vesicles was also marginal, but better than the parent

peptide, thus binding at 100% POPC could be measured, and values obtained for kon, koff, and KD

for pure POPC vesicles were experimental rather than extrapolated. Vesicle compositions of

50:50:2 up to 90:10:2 POPC/POPG/7MC-POPE were used in addition to 100:2 POPC/7MC-

POPE. The collected fluorescence data from these experiments are shown in Figure 12. Various

graphs of KD, kon, and koff as a function of vesicle POPC content show the data fit and values for

100% POPC vesicles, shown in Figures 13 and 14. The kinetic constants for binding and

dissociation are compared for magainin 2 and MG2 in Table 3.

32

Figure 12: MG2 binding data. Curves in (A), (C), (E), and (G) represent raw fluorescence data from binding at 25

µM lipid and 1 µM peptide concentration. Vesicle composition is POPC/POPG/7MC-POPE 50:50:2 in (A), 70:30:2

in (C), 80:20:2 in (E), and 100:2 (POPC:7MC-POPE) in (G). All curves are the average of ~20 curves and the gray

line is the one-exponential fit. To the right of each graph is the corresponding linear regression of the apparent rate

constant to the lipid concentration, yielding kon and koff for each vesicle composition.

33

Figure 13: (A) MG2 on-rate (A) and off-rate (B) constants as a function of POPC content in mixed POPC/POPG

vesicles. The binding experiments were performed with 1µM CE2 and various lipid concentrations from 25 to 200

µM. Increasing the neutral character of vesicles causes marked decrease in the affinity of the peptide for the vesicle.

Linear regression of the data is shown in each graph.

34

Figure 14: MG2 equilibrium dissociation constant as a function of POPC content in POPC/POPG mixed vesicles.

MG2 bound sufficiently well that KD values were experimentally determined for 100% POPC vesicles. The top

graph shows the data plotted using a linear scale. The bottom graph shows the natural log of the data and the linear

correlation in this form. The lines represent the best data fit in each graph.

35

Table 3: Magainin 2 and MG2 On- and Off-Rate Constants and Equilibrium Dissociation Constants

Vesicle composition Peptide Binding kinetics Dissociation constant*

POPC/POPG kon (M-1

s-1

) koff (s-1

) KD (µM)

50:50 Magainin 2

MG2 (9.1 ± 0.4) 10

5

(6.5 ± 0.6) 105

20 ± 9

26 ± 3

22 ± 10

40 ± 6

70:30 Magainin 2

MG2 (8.5 ± 0.7) 10

5

(5.6 ± 0.8) 105

77 ± 14

27 ± 3

91 ± 18

49 ± 9

80:20 Magainin 2

MG2 (7.8 ± 0.7) 10

5

(3.6 ± 0.6) 105

160 ± 15

37 ± 1

205 ± 27

103 ± 17

90:10

100:0‡

Magainin 2

MG2

Magainin 2

MG2

(5.5 ± 0.9) 105

(1.9 ± 0.5) 105

9.7 104

(0.2 ± 0.2) 104

350 ± 20

25 ± 6

600

23 ± 2

640 ± 110

126 ± 45

6000

2000 ± 1200

*KD is calculated solely from binding kinetics and expressed as a function of lipid concentration, not vesicle

concentration. ‡Values for 100% POPC vesicles were extrapolated from the best-fit curves for magainin 2.

36

Carboxyfluorescein Efflux Experiments

Carboxyfluorescein dye (CF) was encapsulated in large unilamellar vesicles at self

quenching concentrations. Mixing peptide with the CF loaded vesicles permeabilized the

vesicles and caused the CF to leak into the surrounding buffer. This dilution of the CF relieved

the self-quenching and the increase in CF fluorescence was measured over time. Peptide

concentration in all experiments was held constant at 1 µM and lipid concentration was varied

from 25 to 200 µM. Variances in technique and instrumentation require definition of ―full

release,‖ and this was established as causing fractional release in the range of ~0.8 to 1.

Early experiments indicated that the fractional release of CF was notably lower than

expected for CE2 compared to cecropin A (13). The initial interaction of CE2 and vesicles was

not being captured in the long runs due to excessive signal averaging. Thus, split timebases were

used in order to capture data more frequently in the initial mixing period, and at longer intervals

afterwards. The result was fractional release at the expected value, or full release. We see in

Figure 15 that CE2 releases all dye from the vesicles; however, at high lipid concentrations, the

efflux takes much longer than native cecropin A. For 50:50 POPC/POPG vesicles, at 25 and 50

µM, the rate of release for CE2 and cecropin A are about the same. At 100 µM, CE2 takes about

600 s to release 80% of the dye where cecropin A only took 400 s to achieve this same release

level. At 200 µM, CE2 takes about 2500 s to invoke 80% release where cecropin A takes about

650 s. But, these differences in efflux rates are minimal when considering potential error

accumulation in these experiments.

37

Figure 15: 1 µM CE2 dye efflux experiments at various concentrations of 50:50 POPC/POPG vesicles. The curves

each represent one experiment with 25, 50, 100, and 200 µM lipid concentrations. The fastest curve is 25 µM and

the slowest curve is 200 µM. Notable is the extremely rapid initial efflux, as well as the induced complete release

(top). There is significant fractional CF release in the short time period immediately following mixing of peptide

and vesicles, and this sharp increase seems to be concentration independent (bottom).

38

MG2 induced complete release of vesicles at 50:50 and 70:30 POPC/POPG but at a much

faster rate than magainin 2. The efflux curves for MG2 can be seen in Figure 16. Using 50:50

POPC/POPG vesicles, MG2 produced 80% release for 25 µM lipid in about 1 second where it

took Magainin 2 about 250 s. At 50 µM lipid for the same composition, it took MG2 about 3

seconds to elicit 80% release where Magainin 2 took 750 s. Using 70:30 POPC/POPG vesicles,

at 25 µM lipid, MG2 produced 80% release in about 10 s and magainin 2 took about 2500 s. At

50 µM lipid, MG2 elicited 80% release in about 20 s, and magainin 2 took about 4000 s.

Changing the lipid composition to 100% POPC caused the efflux rate to diminish greatly, as seen

in Figure 17.

39

Figure 16: 1 µM MG2 dye efflux experiments at various concentrations of 50:50 and 70:30 POPC/POPG vesicles.

The top graph is for 50:50 POPC/POPG and the bottom graph is for 70:30 POPC/POPG. The curves each represent

one experiment with 25, 50, 100, and 200 µM lipid concentrations where the fastest curve is 25 µM and the slowest

curve is 200 µM.

40

Figure 17: 1 µM MG2 dye efflux experiment with 50 µM 100% POPC vesicles. Even with a five hour timescale,

efflux was not complete, showing the significant effect of incorporating negative phospholipids in these vesicles for

MG2.

41

ANTS/DPX Assay

The mechanism of dye release by CE2 was measured using the ANTS/DPX assay.

Vesicles were prepared by incorporating the fluorophore/quencher pair ANTS and DPX. If

release is all-or-none, then the fluorescence quenching inside the vesicles is independent of the

peptide concentration. The plot of quenching inside the vesicles (Qin) as a function of

fluorescence outside the vesicles (fout) for all-or-none peptides is a horizontal line. CE2 causes

graded release where cecropin A causes all-or-none release. MG2 also causes graded release and

magainin-2 causes all-or-none release.

The mechanism of dye release by CE2 and MG2 was determined using the ANTS/DPX

assay and this data is represented in Figures 18 and 19 respectively. Vesicles were prepared by

encapsulating the fluorophore/quencher pair ANTS and DPX. If the mechanism of release is all-

or-none, all the contents of some vesicles is released, and the fluorescence quenching inside the

vesicles is independent of the peptide concentration. Graded release causes the vesicles to leak

part of their contents; however, usually the release of ANTS and DPX is unequal, and the

fluorescence inside the vesicles increases. The plot of quenching inside the vesicles (Qin) as a

function of fluorescence outside the vesicles (fout) for all-or-none peptides is a horizontal line and

for graded peptides the plot is a rising curve. The fit equations used for these experiments are

those established by Ladokhin et al., and shown in Eqs. 9a and 9b (26,27).

(9a)

(9b)

42

and are the fluorescence intensities inside the vesicles without quencher.

represents the initial concentration of DPX which was 8mM, is the portion of ANTS

outside the vesicles, is the dynamic quenching constant, 50 M-1

(26,27), is the static

quenching constant (a fit value from Eq. 9b), and α (also fit from Eq. 9b) is the ratio of DPX

released to ANTS released. Since DPX is positively charged, the incorporation of negatively

charged POPG in the vesicles is likely influencing the shape of the curve relative to previous

experiments. Selective release of DPX is apparent, with an α value of 3 for the MG2 experiment

where 70:30 POPC/POPG vesicles were used, and an α value of 9 for the CE2 experiments

where 50:50 POPC/POPG vesicles were used.

43

Figure 18: ANTS/DPX assay from three experiments for CE2, showing graded mechanism for ~600 µM 50:50

POPC/POPG lipid vesicles with varying concentrations of CE2. If the mechanism were all-or-none, the data would

have generally followed the dashed line. The sigmoidal shape of the curve, governed by the fit parameter α in Eq.

9b, indicates high propensity for selective release of DPX, probably due to the negative character of the membrane.

The fit parameters for Eq. 9b were α = 8.6, and Ka = 188.

44

Figure 19: ANTS/DPX assay for MG2 using ~600 µM 70:30 POPC/POPG vesicles. Again, the sigmoidal shape is

probably attributed to the large amount of POPG in the vesicles. The fit parameters for Eq. 9b were α = 3.1, and Ka

= 192.

45

Circular Dichroism Experiments

The secondary structure of the peptides was determined by circular dichroism, CD, for

mixed compositions of POPC/POPG vesicles. A buffer containing PO43-

, pH 7.5 was used to

dilute the vesicles. Mixed with a high concentration of 50:50 POPC/POPG vesicles, CE2 was

69% and MG2 was 42% helical. Helicities with 100% POPC vesicles at high lipid concentration

were 35% for CE2 and 21% for MG2, but these values are not corrected for the high portion of

unbound peptide. The CD data for CE2 can be seen in Figure 20, and MG2 CD data can be seen

in Figure 21.

46

Figure 20: CD spectra for CE2 using 50:50 POPC/POPG (A) and 100% POPC (B) vesicles.

47

Figure 21: CD spectra for MG2 using 50:50 POPC/POPG (A) and 100% POPC (B) vesicles.

48

Thermodynamics of Peptide-Lipid Interactions

Thermodynamic values for peptide-LUV interactions were calculated as in previous work

in our lab. The experimental values for the Gibbs free energy of binding, ΔGbind(exp), were

calculated using the KD values of CE2 and MG2 in Eq. 10:

ΔGbind(exp) = RT ln KD – 2.4 kcal/mol (10)

Here R is the gas constant, T is the absolute temperature in Kelvin, and the term 2.4 kcal/mol is

the cratic correction for mixing (10, 34). The Gibbs energy of binding to the membrane-water

interface, ΔGif, is calculated using the Wimley-White interfacial scale (35, 36). The binding

process couples the association of the unstructured peptide with the lipid bilayer and the folding

of this peptide into an α-helix (34). Therefore, ΔGif is the sum of two terms: binding in an

unstructured state and folding on the membrane. ΔGif was calculated based on the determined

helicities of the peptides. Estimation of the Gibbs energy of transfer of the peptide from the

membrane interface to the bilayer interior was done based on the whole-residue octanol transfer

scale (37). Admittedly, the bilayer interior is not ideally represented by octanol, but this

estimation is appropriate in this application (38). The complete thermodynamic cycle is given as

the sum of the terms in Eq. 11:

ΔGins = ΔGf + ΔGoct – ΔGif. (11)

Since the value for ΔGf is very small compared to the other terms, we get the relationship in Eq.

12:

ΔGins ≈ ΔGoct – ΔGif = ΔGoct-if. (12)

49

The experimental and theoretical Gibbs energies of binding are mostly the same. Discrepancies

can arise when the peptide may form salt bridges, which increases the stability of the folded

peptide (10). The thermodynamic calculations were performed with Membrane Protein Explorer

(39) and can be seen in Table 4.

50

Table 4: Thermodynamic Parameters for Peptide Binding and Insertion into POPC Bilayers at Room Temperature

Peptide KD*

ΔGbind(exp) (kcal/mol)

ΔGif(calcd) (kcal/mol)

% helix ΔGoct (kcal/mol)

ΔGoct-if

(kcal/mol)

cecropin A 1 mM -6.4 -2.7 70 31.1 33.8

CE2 5 mM -5.5 -4.3 69 31.2 35.5

magainin 2 6 mM -5.4 -6.0 83 20.3 26.3

MG2 2 mM -6.1 -2.8 42 19.3 22.1

*KD values were calculated with only binding kinetics on- and off-rate constants.

51

DISCUSSION

Mechanism of Vesicle Perturbation

One of the most intriguing discoveries in this research was the difference in the

mechanisms by which the mutant peptides disrupt vesicles compared with the parent peptide.

Cecropin A and magainin 2 disrupt vesicles by means of an all-or-none mechanism (13,14,25).

However, from the ANTS/DPX assay, both CE2 and MG2 undergo a graded mechanism. The

exact reason remains elusive, however, there are some hypotheses. The calculated Gibbs free

energy of insertion, ΔGins, for CE2 is 36 kcal/mol using a peptide helicity of 69% upon binding.

Being this high, one would expect an all-or-none mechanism as with the parent peptide.

However, CE2 is unique in that it contains two, separate helical regions. The helix with the

higher hydrophobic moment may be anchoring itself on the surface of the buffer-vesicle

interface and the other helix may be ―dipping‖ into the bilayer, disturbing the integrity of the

vesicle and allowing contents to steadily leak out in graded fashion. One stipulation in

developing an appropriate antimicrobial peptide is the strength of binding. If binding is too

strong, it may hinder the ability of a peptide to translocate or form the appropriate surface defects

in the membrane to cause leakage.

The free energy of insertion for MG2 is 22 kcal/mol, based on a vesicle-bound helicity of

42%. This is near the cutoff value 20 kcal/mol, but is sufficiently close to deem this a ―gray

zone‖ (10). It is difficult to pinpoint the exact reason for the difference in the mechanism.

Magainin 2, with a ΔGins of 26 kcal/mol with 100% POPC vesicles, has a helicity of 83%.

Experimentally, MG2 had a 20% helicity with pure POPC vesicles, however, this is an

uncorrected value not accounting for unbound peptide. Using the estimate of 42% helicity, the

52

ΔGins of 22 kcal/mol is reasonably close to the 20 kcal/mol border and graded release is not

unreasonable.

Kinetics of Binding

The binding observed with CE2 is in line with our estimations. The binding kinetics

were mostly less than a factor of two different. The difference in dissociation constants is one of

the most notable differences, extrapolated to 1mM for cecropin A and 5mM for CE2, but all

dissociation constants were larger for CE2, indicating weaker binding compared to cecropin A.

The dipping event mentioned above for CE2 may be responsible for the deviation of some

kinetic binding traces from one exponential fits. However, without an estimate of the rate of

insertion, or a method to verify if it does indeed happen, we cannot conclude that this is the only

concurrent process affecting the data.

Binding for MG2 is tighter than for magainin 2, with dissociation constants of 2 mM and

6 mM respectively. The difference is about two-fold for most lipid compositions. One reason

MG2 may be binding better is because the off-rate constant is independent of the negative lipid

character of the vesicles (Figure 13). Another consideration is the differences in helicity of the

native and modified peptides. Magainin 2 has a helicity of about 56% when bound to 50:50

POPC/POPG membranes where MG2 only has a helicity of 42% to the same vesicle

composition. This affects the values obtained for the Gibbs free energy of insertion for the

peptides.

Thermodynamics of Binding

Peptide mechanism does not appear to be limited by thermodynamic constraints only. The Gibbs

free energy of insertion of 36 kcal/mol for CE2 suggests that it should operate with an all-or-

53

none mechanism. Although the complete release in efflux experiments support this, the data

from the ANTS/DPX assay imply that CE2 is a graded release peptide.

The effect of helicity on the calculated free energies is also important going forward in

novel peptide development. The markedly decreased helicity of MG2 compared with magainin 2

shifted the Gibbs energy of insertion into the ―gray zone,‖ making it difficult to pinpoint the

differences in the peptides.

Peptide Efflux

Cecropin A and CE2 both induce complete release of vesicle contents, independent of the

difference in mechanisms. Although the rate of release differs slightly for higher concentrations

of lipid in CE2, they still match reasonably well to the rates of release for cecropin A.

Both magainin 2 and MG2 induce complete release, but MG2 efflux is about 250 times

faster. It is interesting that a graded release by MG2 also produces complete release (one would

expect efflux to cease once the mass imbalance dissipated).

Final Thoughts

Although binding is similar for both peptides, the lowered ΔGins for MG2 is probably

responsible for the change in mechanism compared with magainin 2. However, this does not

explain the complete release seen in efflux experiments. Also, it is intriguing that MG2releases

vesicle contents so much faster than magainin 2, even though it has a much lower helicity.

For CE2 the ΔGins is well outside the region where one would expect a graded release

mechanism, and again, the complete release observed is counterintuitive. Another interesting

point is the CE2 on-rate constant dependence on POPC content in vesicles. One might expect an

54

exponential decrease in the on-rate as POPG content is decreased in the vesicles. It appears there

is a sharp transition where the kon decreases between 70 and 80% POPC content.

Studies of peptide translocation and pore states may help confirm the mechanisms

determined for CE2 and MG2. Design and analysis of helical properties of the peptides and the

effects on peptide effectiveness may also warrant investigation.

55

FUTURE WORK

In order to verify whether peptides which undergo all-or-none mechanisms, it would be

necessary to perform experiments which test for translocation of the peptides. If an all-or-none

peptide were to translocate, it could produce graded results and may cease efflux at a certain

level by relieving the mass imbalance across the membrane. Further, testing lipid flip-flop rates

and their effects on the translocation or lack thereof would also support the determined

mechanisms of MG2 and CE2.

Another means to verify the results of the thermodynamic values calculated in this

experiment would be through differential scanning calorimetry and isothermal titration

calorimetry. These methods could support or refute the results of this research based on the

obtained energy values for the binding of these peptides.

Analysis of the pore state lifetimes would also help verify the mechanism for these

peptides. If the pores are long-lived, it would support the all-or-none mechanism and if they are

short-lived, a graded mechanism would be indicated. This would help to confirm that the

mechanism of release for CE2 is indeed graded, and not the results of another, unknown process.

56

ABBREVIATIONS

POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-

glycero-3-phospho-(1’-rac-glycerol); POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphoethanolamine; 7MC, 7-methoxycoumarin-3-carboxylic acid; ANTS, 8-

aminonaphthalene-1,3,6-trisulfonic acid; DPX, p-xylene-bis-pyridinium bromide; LUV, large

unilamellar vesicle; FRET, fluorescence resonance energy transfer; CD, circular dichroism;

MPEx, Membrane Protein Explorer; ΔGif, Gibbs energy of peptide binding to membrane-water

interface as a helix; ΔGins, Gibbs energy of insertion from interface into membrane; ΔGbind,

experimentally determined Gibbs energy of binding; ΔGf, Gibbs energy of folding to an α-helix

in water; ΔGoct, Gibbs energy of peptide transfer from water to octanol; ΔGoct-if = ΔGoct - ΔGif;

kon, on-rate constant; koff, off-rate constant; KD, equilibrium dissociation constant.

57

REFERENCES

1. Hultmark, D., Steiner, H., Rasmuson, T., and Boman, H. (1980) Insect immunity.

Purification and properties of three inducible bactericidal proteins from hemolymph of

immunized pupae of Hyalophora cecropia. Eur. J. Biochem. 106, 7-16.

2. Harder, J., Gläser, R., and Schröder, J. (2007) Human antimicrobial proteins – Effectors of

innate immunity. J. Endotoxin Research 13, 317-339.

3. Hancock, R., and Chapple, D. (1999) Peptide antibiotics. Antimicrob. Agents Chemother. 43,

1317–1323.

4. Tossi, A., Sandri, L., and Giangaspero, A. (2000) Amphipathic, alpha-helical antimicrobial

peptides. Biopolymers 55, 4-30.

5. Jenssen, H., Hamill, P., and Hancock, R. (2006) Peptide antimicrobial agents. Clin.

Microbiol. Rev. 19, 491-511.

6. Papo, N., and Shai, Y. (2003) Can we predict biological activity of antimicrobial peptides

from their interactions with model phospholipid membranes? Peptides 24, 1693-1703.

7. Hancock, R., and Diamond, G. (2000) The role of cationic antimicrobial peptides in innate

host defenses. Trends Microbiol. 8, 402-410.

8. Hoskin, D., and Ramamoorthy, A. (2007) Studies on anticancer activities of antimicrobial

peptides. Biochim. Biophys. Acta. 1778, 357-375.

9. Shai, Y. (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66,

236-248.

10. Almeida, P. F. F., and Pokorny, A. (2009) Mechanisms of antimicrobial, cytolytic and cell-

penetrating peptides: From kinetics to thermodynamics. Biochemistry 48, 8083-8093.

11. Hancock, R. (1997) Peptide antibiotics. Lancet. 349, 418-422.

12. RasMol Molecular Graphics Visualization Tool. (2009) http://www.openrasmol.org/

13. Gregory, S., Cavenaugh, A., Journigan, V., Pokorny, A., and Almeida, P. F. F. (2008) A

quantitative model for the all-or-none permeabilization of phospholipids vesicles by the

antimicrobial peptide cecropin A. Biophys. J. 94, 1667-1680.

14. Yandek, L., Pokorny, A., Florén, A., Knoelke, K., Langel, Ü., and Almeida, P. F. F. (2007)

Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers. Biophys.

J. 92, 2434-2444.

58

15. Ehrenstein, G., and Lehar, H. (1977) Electrically gated ionic channels in lipid bilayers. Q.

Rev. Biophys. 10, 1-34.

16. Ludtke, S., He, K., Heller, W., Harroun, T., Yang, L., and Huang, H. (1996) Membrane

pores induced by magainin. Biochemistry 35, 13723-13728.

17. Matsuzaki, K., Murase, N., Fujii, N., and Miyajima, K. (1996) An antimicrobial peptide,

magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and rapid

translocation. Biochemistry 35, 11361-11368.

18. Pokorny, A., and Almeida, P. F. F. (2004) Kinetics of dye efflux and lipid flip-flop induced

by δ-lysin in phosphatidylcholine vesicles and the mechanism of graded release by amphipathic,

α-helical peptides. Biochemistry 43, 8846-8857.

19. Pokorny, A., Birbeck, T., and Almeida, P. F. F. (2002) Mechanism and kinetics of δ-lysin

interaction with phospholipid vesicles. Biochemistry 41, 11044-11056.

20. Pokorny, A., and Almeida, P. F. F. (2005) Permeabilization of raft-containing lipid vesicles

by δ-lysin: A mechanism for cell sensitivity to cytotoxic peptides. Biochemistry 44, 9538-9544.

21. Steiner, H., Hultmark, D., Engstrom, A., Bennich, H., and Boman, H. G. (1981) Sequence

and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246-248.

22. Steiner, H. (1982) Secondary structure of the cecropins: Antibacterial peptides from the

moth Hyalophora cecropia. FEBS Lett. 137, 283-287.

23. Holak, T. A., Engstrom, A., Kraulis, P. J., Lindeverg, G., Bennich, H., Jones, A.,

Gronenborn, A. M., and Clore, G. M. (1988) The solution conformation of the antibacterial

peptide cecropin A: A nuclear magnetic resonance and dynamical simulated annealing study.

Biochemistry 27, 7620-7629.

24. Zasloff, M. (1987) Magainins, a class of antimicrobial peptides from Xenopus skin:

Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc.

Natl. Acad. Sci. 84, 5449-5453.

25. Gregory, S., Pokorny, A., and Almeida, P. F. F. (2009) Magainin 2 revisited: A test of the

quantitative model for the all-or-none permeabilization of phospholipid vesicles. Biophys. J. 96,

116-131.

26. Ladokhin, A., Wimley, W., and White, S. (1995) Leakage of membrane vesicle contents:

Determination of mechanism using fluorescence requenching. Biophys. J. 69, 1964-1971.

27. Ladokhin, A., Wimley, W., Hristova, K., and White, S. (1997) Mechanism of leakage of

contents of membrane vesicles determined by fluorescence requenching. Methods Enzymol. 278,

474-486.

59

28. Bartlett, G. (1959) Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466-

468.

29. Vaz, W., and Hallman, D. (1983) Experimental evidence against the applicability of the

Saffman-Delbrück model to the translational diffusion of lipids in phosphatidylcholine bilayer

membranes. FEBS Lett. 152, 287-290.

30. Kates, M. (1972) Techniques in Lipidology. In Laboratory Techniques in Biochemistry

and Molecular Biology. Work, T. and Work. E., Eds. North-Holland Publishing, Amsterdam,

Netherlands.

31. Ladokhin, A., and White, S. (1999) Folding of amphipathic α-helices on membranes:

Energetics of helix formation by mellitin. J. Mol. Biol. 285, 1363-1369.

32. Wieprecht, T., Apostolov, O., Beyermann, M., and Seelig, J. (1999) Thermodynamics of

the α-helix-coil transition of amphipathic peptides in a membrane environment: Implications for

the peptide-membrane binding equilibrium. J. Mol. Biol. 294, 785-794.

33. Chen, Y., Yang, T., and Martinez, H. (1972) Determination of the secondary structures of

proteins by circular dichroism and optical rotary dispersion. Biochemistry 11, 4120-4131.

34. White, S., and Wimley, W. (1999) Membrane protein folding and stability: physical

principles. Annu. Rev. Biophys. Biomol. Struct. 28, 319-365.

35. Wimley, W., and White, S. (1996) Experimentally determined hydrophobicity scale of

proteins at membrane interfaces. Nat. Struct. Biol. 3, 842-848.

36. Hristova, K., and White, S. (2005) An experiment-based algorithm for predicting the

partitioning of unfolded peptides into phosphatidylcholine bilayer interfaces. Biochemistry 44,

12614-12619.

37. Wimley, W., Creamer, T., and White, S. (1996) Solvation energies of amino acid side

chains and backbone in a family of host-guest pentapeptides. Biochemistry 35, 5109-5124.

38. Jayasinghe, S., Hristova, K., and White, S. (2001) Energetics, stability, and prediction of

transmembrane helices. J. Mol. Biol. 312, 927-934.

39. Jaysinghe, S., Hristova, K., Wimley, W., Snider, C., and White, S. (2009)

http://blanco.biomol.uci.edu/MPEx.